Figure 1. Design and activity analysis of native FGF2 and its variants. (A ) Based on the crystal structure of FGF2 (PDB ID: 1FQ9), FGF2-FGFR1-heparin complex structure (left panel), close-up view of two surface-exposed residues, Lys129 and Phe17, near the heparin- and receptor-binding regions, respectively, and two surface-exposed cysteines (Cys69 and Cys87) on FGF2 (right panel). (B ) Schematic representation of two FGF2 variants, including FGF2C69A (1 ) and FGF2C87A(2 ). The residues labeled in green indicate mutation sites, and the residues marked in red are single conjugation sites for each FGF2 variant. (C ) Immunoblotting analysis of p-ERK1/2 and ERK1/2 levels in cell lysates of HUVECs after treatment with compounds 1 , 2 (10 ng·mL-1). ERK was used as a loading control (n = 3). (E ) Schematic representation of another two synthetic FGF2 variants, including FGF2C69A/C87A/F17C (3 ) and FGF2C69A/C87A/K129C (4 ). (F ) Immunoblotting analysis of p-ERK1/2 and ERK1/2 levels in cell lysates of HUVECs after treatment with compound 3 , 4 (10 ng·mL-1) (n = 3). (D, G ) Quantitative analysis of p-ERK1/2 in the indicated groups. Data are displayed as means ± SEM. **P < 0.01,***P < 0.001 vs. control group;#P < 0.05 vs. FGF2 group; n.s., no significance.